1. Field of the Invention
The invention relates generally to methods of activating the Rse tyrosine kinase receptor. More particularly, the invention relates to methods of enhancing survival, proliferation and/or differentiation of cells comprising the Rse receptor (such as glial cells) using gas6. The invention also relates to gas6 variants, particularly those which are less .gamma.-carboxylated than gas6 isolated from nature.
2. Description of Related Art
Specific signals that control the growth and differentiation of cells in developing and adult tissues often exert their effects by binding to and activating cell surface receptors containing an intrinsic tyrosine kinase activity. Mark et al. recently described the human and murine complementary DNA sequences of the receptor tyrosine kinase Rse that is preferentially expressed in the adult brain (Mark et al., J. Biol. Chem. 269:10720 [1994]). The extracellular domain of Rse receptor is composed of two immunoglobulin-like (Ig-L) repeats followed by two fibronectin type III repeats. Complementary DNA sequences encoding proteins identical to human (Ohashi et al., Oncogene 9:699 [1994]) and murine Rse (Lai et al, Oncogene 9:2567 [1994]) have been reported independently, and termed Sky and Tyro3, respectively. See also Fujmimoto and Yamamoto Oncogene 9: 693 (1994) concerning the murine equivalent to Rse they call brt and Dai et al. Oncogene 9: 975 (1994) with respect to the human molecule they call tif.
The expression of Rse in various tissues has been investigated. Lai et al., supra, found that, in the adult brain, Rse mRNA is localized in neurons of the neocortex, cerebellum and hippocampus. Schulz et al. similarly found that Rse is expressed at high levels in the cerebral cortex, the lateral septum, the hippocampus, the olfactory bulb and in the cerebellum. The highest levels of Rse expression in the brain were found to be associated with neurons. (Schulz et al. Molec. Brain Res. 28: 273-280 [1995]). In the central nervous system (CNS) of mice, the expression of Rse is detected at highest levels during late embryonic stages and post birth, coincident with the establishment and maintenance of synaptic circuitry in cortical and hippocampal neurons (Lai et al., supra and Schneider et al., Cell 54:787-793 [1988]). This process is believed to be regulated locally, by cells that are in direct contact or positioned close to one another. By Northern blot analysis, Mark et al., supra, found that high levels of Rse mRNA were present in samples of RNA from the brain and kidney. Dai et al., supra found that Rse was highly expressed in human ovary and testes. The expression of Rse in various human cell lines was also analyzed by Mark et al., supra. Little, or no, Rse mRNA was detected by Northern blotting of mRNA samples from the monocyte cell line THP-1 or the lymphoblast-like RAJl cells. However, the Rse transcript was detected in a number of hematopoietic cell lines, including cells of the myeloid (i.e., myelogenous leukemia line K562 and myelomonocytic U937 cells) and the megakaryocytic leukemia lines DAMI and CMK11-5, as well as the human breast carcinoma cell line MCF-7. In the cell lines examined, the highest level of expression was observed in Hep 3B cells, a human hepatocarcinoma cell line.
Rse is structurally related to Axl (also known as Ufo or Ark) and shares 43% overall amino acid sequence identity with this tyrosine kinase receptor. See O'Bryan et al., Mol. Cell. Biol. 11:5016 (1991), Janssen et al., Oncogene 6:2113 (1991), Rescigno et al. Oncogene 5:1908 (1991) and Bellosta et al. 15: 614 (1995) concerning Axl. Rse and Axl, together with c-Mer (Graham et al., Cell Growth Differ. 5:647 [1994]), define a class of receptor tyrosine kinases whose extracellular domains resemble neural cell recognition and adhesion molecules (reviewed by Ruitishauser, U. in Current Opin. Neurobiology 3:709 [1993] and Brummendorf and Rathjen in J. Neurochemistry 61:1207 [1993]). Like Rse, Axl is also expressed in the nervous system, but is more widely expressed than Rse in peripheral tissues.
Putative ligands for the Rse and Axl receptors have been reported. Varnum et al. Nature 373:623 (1995) and Stitt et al. Cell 80: 661-670(1995) recently reported that gas6 (for growth arrest-specific gene 6) is a ligand for Axl. Gas6 belongs to a set of murine genes which are highly expressed during serum starvation in NIH 3T3 cells (Schneider et al., Cell 54:787-793 [1988]). These genes were designated growth arrest-specific genes, since their expression is negatively regulated during growth induction. The human homolog of murine gas6 was also cloned and sequenced by Manfioletti et al. in Molec. Cell Biol. 13(8):4976-4985 (1993). They concluded that gas6 is a vitamin K-dependent protein and speculated that it may play a role in the regulation of a protease cascade relevant in growth regulation. Gas6 is expressed in a variety of tissues including the brain. See also Colombo et al. Genome 2:130-134 (1992) and Ferrero et al. J. Cellular Physiol. 158:263-269(1994) concerning gas6.
Stitt et al., supra further reported that protein S is the ligand for Tyro3. Protein S is a vitamin K-dependent plasma protein that functions as an anticoagulant by acting as a cofactor to stimulate the proteolytic inactivation of factors Va and VIIIa by activated protein C. Reviewed in Easmon et al. Aterioscler. Thromb. 12:135 (1992). Accordingly, protein S is an important negative regulator of the blood-clotting cascade. See Walker et al., J. Biol. Chem. 255:5521-5524 (1980), Walker et al., J. Biol. Chem. 256:11128-11131 (1981), Walker et al., Arch. Biochem. Biophys. 252: 322-328 (1991), Griffin et al. Blood 79: 3203 (1992) and Easmon, D., Aterioscler. Thromb. 12:135 (1992). The discovery that about half of the protein S in human plasma is bound to C4BP further supports the notion that protein S is involved in the complement cascade. Dahlback et al., PNAS(U.S.A.) 78: 2512-2516(1981). The role of protein S as a mitogen for smooth muscle cells has also been reported. Gasic et al., PNAS (U.S.A.) 89:2317-2320(1992).
Protein S can be divided into four domains (see FIGS. 1A, 1C and 1D herein). Residues 1-52 (Region A) are rich in .gamma.-carboxyglutamic acid (Gla) residues which mediate the Ca.sup.2+ dependent binding of protein S to negatively charged phospholipids (Walker, J. Biol. Chem. 259:10335 [1984]). Region B includes a thrombin-sensitive loop. Region C contains four epidermal growth factor (EGF)-like repeats. Region D is homologous to the steroid hormone binding globulin (SHBG) protein (Hammond et al., FEBS Lett. 215:100 [1987]). As discussed by Joseph and Baker (FASEB J. 6:2477 [1994]), this region is homologous to domains in the A chain of laminin (23% identity) and merosin (22% identity) and to a domain in the Drosophila crumbs (19%).
Murine and human gas6 cDNAs encode proteins having 43 and 44% amino acid sequence identity respectively to human protein S.